Despite advances in the development of complex culture technologies, the utility, survival, and function of large 3D cell aggregates, or spheroids, are impeded by mass transport limitations. The incorporation of engineered microparticles into these cell aggregates offers a promising approach to increase spheroid integrity through the creation of extracellular spaces to improve mass transport. In this study, we describe the formation of uniform oxygenating fluorinated methacrylamide chitosan (MACF) microparticles via a T-shaped microfluidic device, which when incorporated into spheroids increased extracellular spacing and enhanced oxygen transport via perfluorocarbon substitutions. The addition of MACF microparticles into large liver cell spheroids supported the formation of stable and large spheroids (>500 μm in diameter) made of a heterogeneous population of immortalized human hepatoma (HepG2) and hepatic stellate cells (HSCs) (4 HepG2/1 HSC), especially at a 150:1 ratio of cells to microparticles. Further, as confirmed by the albumin, urea, and CYP3A4 secretion amounts into the culture media, biological functionality was maintained over 10 days due to the incorporation of MACF microparticles as compared to controls without microparticles. Importantly, we demonstrated the utility of fluorinated microparticles in reducing the number of hypoxic cells within the core regions of spheroids, while also promoting the diffusion of other small molecules in and out of these 3D in vitro models.

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candidates, and in the past preclinical animal models have been employed that do not necessarily provide results that translate into humans. [2] It has been shown that beginning drug discovery in human cell-based platforms can decrease failure rates. [3] This is only one illustrative example highlighting the usefulness of humanized cell-based platforms in the study of human health and disease. Emerging technologies such as microphysical systems, organs-on-a-chip, 3D bioprinting, and cell spheroid/organoids offer tantalizing utility for medicine. [4] Human cell-based organoids specifically present more realistic and physiologically relevant models, [5] and provide significant utility for personalized disease modelling, drug screening, and even regenerative medicine research. [3] Organoids are miniaturized and simplified versions of organs typically derived from stem cells. They possess threedimensional microanatomy with several organ-specific differentiated and progenitor cell types. Multi-cellular spheroids present a simplified version of organoids using one organ-specific cell-type to reduce the overall complexity and are often the preferred starting point for organoid research. [6] A persistent and significant challenge for organoids and spheroids alike is maintaining sufficient oxygen perfusion to create large anatomically relevant functional tissues in vitro. [7] Past attempts to artificially improve oxygen transport includes artificial microvessels, direct fluid perfusion, and oxygen vesicles for oxygen transport. [8] Each of these techniques faces their own challenges, and despite the efforts invested in developing them, insufficient oxygenation challenges persist ultimately limiting the size of organoids/ spheroids that can be produced, the ability to maintain them long-term, and consequently their physiologic relevance to medical applications. In this study we report a novel strategy to enhance oxygen transport in 3D in vitro human cell cultures using chitosan-PFC oxygenating microgels added to cell aggregate spheroids, enabling the ability to tune local spheroid oxygenation to enhance spheroid utility. One important advantage of this approach is that it combines several mechanisms to boost oxygen transport from the surrounding environment. First, Poor oxygen transport is a major obstacle currently for 3D microtissue culture platforms, which at this time cannot be grown large enough to be truly physio logically relevant and replicate adult human organ functions. To overcome internal oxygen transport deficiencies, oxygenating microgels are formed utilizing perfluorocarbon (PFC) modified chitosan and a highly scalable water-in-oil miniemulsion method. Microgels that are on the order of a cell diameter (≈10 µm) are formed allowing them to directly associate with cells when included in 3D spheroid culture, while not being internalized. The presence of immobilized PFCs in these microgels allows for enhancement and tuning of oxygen transport when incorporated into cultured microtissues. As such, it is demonstra...

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